PROJECT SUMMARY/ABSTRACT With advancing reproductive age, there is a loss of ovarian function and fluctuations in sex steroids that contribute to a greater susceptibility of vascular dysfunction. Importantly, impairments in endothelial function have been demonstrated before menopause, and specifically in the early peri-menopause transition. However, the mechanisms underlying this decline in endothelial function are not known. The purpose of this R01 is to examine key mechanisms contributing to impaired endothelial function in women advancing across reproductive ages, and in particular, the interactions between endothelin-1 (ET-1) and estradiol (E2). The overall hypothesis is that activation of ET-1 and a loss of ETB mediated dilation play a primary role in contributing to impaired endothelial function with advancing reproductive age, and that E2 administration during peri-menopause will attenuate these responses. Accordingly, the first aim of this project will test the hypothesis that ET-1 contributes to impaired endothelial function with advancing reproductive age. The second aim of this project will test the hypothesis that alterations in ETB receptors contribute to impaired endothelial function with advancing reproductive age. The third aim will test the hypothesis that E2 modulates ET-1 receptor control of endothelial function. We will use a cross-sectional design to measure macro- and micro- vascular endothelial-dependent dilation in premenopausal, early peri-menopausal, late peri-menopausal, and postmenopausal women (classified based on reproductive age) under control conditions and in response to ETB receptor and ETA receptor blockade. In addition, we will perform venous endothelial cell biopsies to assess intracellular ET-1 protein expression and ETB receptor expression in endothelial cells. Aim 3 will expand on these cross-sectional comparisons by using a controlled hormone intervention to better isolate the effects of E2 on endothelial function and ET-1 receptor responses in early and late peri-menopausal women. This comprehensive assessment of the ET-1 system will provide novel information on the mechanisms contributing to vascular dysfunction in women, and the impact of E2 therapy on these pathways at specific time points across the menopausal transition. Given the recent controversy surrounding hormone therapy in women, understanding the mechanisms contributing to impaired endothelial function with advancing reproductive age is of both physiological and clinical importance to identify an appropriate time frame for intervention and to optimize the benefits of hormone therapy.